Malaria is a widespread disease in most subtropical countries. It is acquired by infection with a malaria parasite. The socioeconomic impact of this disease is enormous. Malaria exists in different forms, caused by different parasites. The symptoms vary considerably between the different forms.
Plasmodium vivax and Plasmodium falciparum are the two most important human malaria parasites. Other human malaria parasites are Plasmodium ovale and Plasmodium malariae, but these two species are less pathogenic than P. vivax and P. falciparum. P. vivax causes less mortality than P. falciparum. Treatment of P. falciparum is becoming more complicated, because chloroquine-resistant P. falciparum parasites are spreading rapidly and multidrug-resistant parasites have also developed. In addition, chloroquine-resistant P. vivax has been detected, indicating similar problems in the treatment of P. vivax as for P. falciparum. 
At present, there is essentially no effective vaccine available against malaria, at least not for use in humans. Accumulated data, including that from nonhuman primate1,2 and rodent studies,3,4 have indicated that the apical membrane antigen-1 (AMA-1) family of molecules is a target for protective immune responses. In all Plasmodium species reported to date, with the exception of P. falciparum5 and P. reichenowi6 that form a phylogenetic Glade distinct from other malaria parasites, AMA-1 is synthesized de novo as a 66 kDa transmembrane protein. The protein contains a predicted N-terminal signal sequence, an ectodomain, a predicted transmembrane region and a C-terminal cytoplasmic domain. The ectodomain is further divided into three domains (domain I, II and III) defined by disulfide bonds.7 In P. falciparum and P. reichenowi, the protein is expressed as an 83 kDa protein having an N-terminal extension as compared to the 66 kDa forms, referred to as the prosequence. Intraspecies sequence polymorphism due to point mutations8,9,10 reveals clustering of mutations in particular domains of the molecule. Despite this, between species there is considerable conservation of primary amino acid structure and predicted secondary structure. Evidence to date indicates that protection invoked by AMA-1 is directed at conformational epitopes1, 3, 4, 11 located in the AMA-1 ectodomain. Immunization with reduced AMA-1 fails to induce parasite inhibitory antibodies, 3, 11 and so far only those monoclonal antibodies that recognize reduction-sensitive conformational AMA-1 epitopes have been shown to inhibit parasite multiplication in vitro for P. knowlesi12, 13 and P. falciparum.6, 14 This indicates that for an AMA-1 vaccine, the correct conformation will be critical.
Recombinant expression of P. falciparum AMA-1 (Pf AMA-1) in a conformationally relevant way that allows production of clinical grade material has been notoriously difficult. One characteristic important for recombinant expression techniques is the unusually high A+T content of P. falciparum codons in comparison to most other organisms and, in particular, in comparison to most other organisms generally used for recombinant protein expression. The group of Prof. Anders (WEHI, Australia) has developed expression of the ectodomain in E. coli, followed by a refolding protocol, but scaling up this process to levels that allow production of clinical grade material has proven cumbersome. Because eukaryotic expression systems are likely to produce material with the correct disulphide bonds directly, we have focused upon expression in such systems. Expression of the full-length 622 amino acids long Pf AMA-1 protein (7G8 strain) in insect cells using recombinant baculovirus resulted in expression on the surface of insect cells.15 The protein migrated in SDS-PAGE more slowly than the native molecule, indicating glycosylation. Expression in the presence of tunicamycin confirmed this. The Pf AMA-1 protein was used to raise rat monoclonal antibodies (mAbs), some of which could block parasite multiplication in an in vitro assay. These functional mAbs recognized a conformational epitope located in the ectodomain of Pf AMA-1. Reactivity with these mAbs, especially with mAb 4G2, is used as one assay for proper folding of recombinant Pf AMA-1. Relatively low expression levels did not allow the baculovirus system to be developed for the production of clinical grade material.
We have obtained high-level expression of P. vivax AMA-1 (Pv AMA-1) ectodomain in the methylotrophic yeast Pichia pastoris.16 However, this expression system is not likewise suitable to produce a secreted ectodomain of Pf AMA-1. Using the same expression vector as has successfully been used for Pv AMA-1, recombinant Pf AMA-1 P. pastoris clones do not express Pf AMA-1 ectodomain at any level. Analysis of total RNA extracted from induced cultures revealed only truncated mRNA products for Pf AMA-1, so no effective expression of Pf AMA-1 was possible until the present invention. This was a problem because expression of homogeneous Pf AMA-1 in high amounts is highly desirable. Efficient production of Pf AMA-1 gives possibilities to develop a diagnostics or a vaccine and/or a medicine against P. falciparum and/or other Plasmodium species. Presently, such a vaccine or medicine is not available.